Derivatives of [6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-propionamide

ABSTRACT

Derivatives of [6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-propionamide which exhibit good inhibitory effect upon the interaction of CAMs and Leukointegrins and are thus useful in the treatment of inflammatory disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/686,073, filed Oct. 15, 2003, which claims the benefit of U.S.Provisional Application Ser. No. 60/422,446, filed Oct. 30, 2002, andsaid applications are herein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates generally to a class of derivatives of[6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-propionamide,the synthesis of these compounds, their use in the treatment ofinflammatory disease, and pharmaceutical compositions comprising thesecompounds.

2. Background Information

Research spanning the last decade has helped to elucidate the molecularevents attending cell-cell interactions in the body, especially thoseevents involved in the movement and activation of cells in the immunesystem. See generally, Springer, T. Nature, 1990, 346, 425-434. Cellsurface proteins, and especially the Cellular Adhesion Molecules(“CAMs”) and “Leukointegrins”, including LFA-1, MAC-1 and gp150.95(referred to in WHO nomenclature as CD18/CD11a, CD18/CD11b, andCD18/CD11c, respectively) have correspondingly been the subject ofpharmaceutical research and development having as its goal theintervention in the processes of leukocyte extravasation to sites ofinjury and leukocyte movement to distinct targets. For example, it ispresently believed that prior to the leukocyte extravasation, which is amandatory component of the inflammatory response, activation ofintegrins constitutively expressed on leukocytes occurs and is followedby a tight ligand/receptor interaction between integrins (e.g., LFA-1)and one or several distinct intercellular adhesion molecules (ICAMs)designated ICAM-1, ICAM-2, ICAM-3 or ICAM-4 which are expressed on bloodvessel endothelial cell surfaces and on other leukocytes. Theinteraction of the CAMs with the Leukointegrins is a vital step in thenormal functioning of the immune system. Immune processes such asantigen presentation, T-cell mediated cytotoxicity and leukocyteextravasation all require cellular adhesion mediated by ICAMsinteracting with the Leukointegrins. See generally Kishimoto, T. K.;Rothlein; R. R. Adv. Pharmacol. 1994, 25, 117-138 and Diamond, M.;Springer, T. Current Biology, 1994, 4, 506-517.

A group of individuals has been identified which lack the appropriateexpression of Leukointegrins, a condition termed “Leukocyte AdhesionDeficiency” (Anderson, D. C.; et al., Fed. Proc. 1985, 44, 2671-2677 andAnderson, D. C.; et al., J. Infect. Dis. 1985, 152, 668-689). Theseindividuals are unable to mount a normal inflammatory and/or immuneresponse(s) due to an inability of their cells to adhere to cellularsubstrates. These data show that immune reactions are mitigated whenlymphocytes are unable to adhere in a normal fashion due to the lack offunctional adhesion molecules of the CD18 family. By virtue of the factthat LAD patients who lack CD18 cannot mount an inflammatory response,it is believed that antagonism of CD18, CD11/ICAM interactions will alsoinhibit an inflammatory response.

It has been demonstrated that the antagonism of the interaction betweenthe CAMs and the Leukointegrins can be realized by agents directedagainst either component. Specifically, blocking of the CAMs, such asfor example ICAM-1, or the Leukointegrins, such as for example LFA-1, byantibodies directed against either or both of these moleculeseffectively inhibits inflammatory responses. In vitro models ofinflammation and immune response inhibited by antibodies to CAMs orLeukointegrins include antigen or mitogen-induced lymphocyteproliferation, homotypic aggregation of lymphocytes, T-cell mediatedcytolysis and antigen-specific induced tolerance. The relevance of thein vitro studies are supported by in vivo studies with antibodiesdirected against ICAM-1 or LFA-1. For example, antibodies directedagainst LFA-1 can prevent thyroid graft rejection and prolong heartallograft survival in mice (Gorski, A.; Immunology Today, 1994, 15,251-255). Of greater significance, antibodies directed against ICAM-1have shown efficacy in vivo as anti-inflammatory agents in humandiseases such as renal allograft rejection and rheumatoid arthritis(Rothlein, R. R.; Scharschmidt, L., in: Adhesion Molecules, Wegner, C.D., Ed., 1994, 1-8; Cosimi, C. B., et al., J. Immunol. 1990, 144,4604-4612 and Kavanaugh, A. et al., Arthritis Rheum. 1994, 37, 992-1004)and antibodies directed against LFA-1 have demonstratedimmunosuppressive effects in bone marrow transplantation and in theprevention of early rejection of renal allografts (Fischer, A.; et al.,Lancet, 1989, 2, 1058-1060 and Le Mauff, B.; et al., Transplantation,1991, 52, 291-295).

It has also been demonstrated that a recombinant soluble form of ICAM-1can act as an inhibitor of the ICAM-1 interaction with LFA-1. SolubleICAM-1 acts as a direct antagonist of CD18,CD11/ICAM-1 interactions oncells and shows inhibitory activity in in vitro models of immuneresponse such as the human mixed lymphocyte response, cytotoxic T cellresponses and T cell proliferation from diabetic patients in response toislet cells (Becker, J. C.; et al., J. Immunol. 1993, 151, 7224 andRoep, B. O.; et al., Lancet, 1994, 343, 1590).

Thus, the prior art has demonstrated that large protein molecules whichantagonize the binding of the CAMs to the Leukointegrins havetherapeutic potential in mitigating inflammatory and immunologicalresponses often associated with the pathogenesis of many autoimmune orinflammatory diseases. However proteins have significant deficiencies astherapeutic agents, including the inability to be delivered orally andpotential immunoreactivity which limits the utility of theses moleculesfor chronic administration. Furthermore, protein-based therapeutics aregenerally expensive to produce.

It follows that small molecules having the similar ability as largeprotein molecules to directly and selectively antagonize the binding ofthe CAMs to the Leukointegrins would make preferable therapeutic agents.

Several small molecules have been described in the literature thataffect the interaction of CAMs and Leukointegrins. For example, U.S.Pat. No. 6,355,664 and the corresponding WO 98/39303 disclose a class ofsmall molecule, having a hydantoin core, that are inhibitors of theinteraction of LFA-1 and ICAM-1. Of greater relevance to the presentinvention is WO 01/07440 A1, which discloses compounds that instead havean 6,7-dihydro-5H-imidazo[1,2-a]imidazole core. While the compounds thatare specifically described by WO 01/07440 A1 have a more potentinhibitory affect upon the interaction of CAMs and Leukointegrins thando the hydantoins of U.S. Pat. No. 6,355,664 and the correspondingWO9839303, they nevertheless are not ideal therapeutic agents becausethe rate at which they are metabolized is undesirably high.

Thus, the problem to be solved by the present invention is to find smallmolecules that have not only good inhibitory effect upon the interactionof CAMs and Leukointegrins but that also are metabolized at a rate thatis not overly rapid.

BRIEF SUMMARY OF THE INVENTION

The invention is a subset or selection of the6,7-dihydro-5H-imidazo[1,2-a]imidazoles that are generically but notspecifically described by WO 01/07440 A1. Quite surprisingly, thecompounds included within the invention exhibit not only good inhibitoryeffect upon the interaction of CAMs and Leukointegrins but aremetabolized much more slowly than are the compounds that arespecifically described by WO 01/07440 A1. The compounds of the inventionsolve the problem of overly rapid metabolism.

DETAILED DESCRIPTION OF THE INVENTION

The invention comprises compounds of the formula I

wherein:

-   -   R¹ is straight or branched alkyl of 1 to 3 carbon atoms which is        optionally mono- or disubstituted with moieties independently        selected from the group consisting of:        -   (i) oxo and        -   (ii) morpholino;    -   R² and R³ are each, independently selected from the group        consisting of:        -   (A) hydrogen, and        -   (B) straight or branched alkyl of 1 to 4 carbon atoms which            alkyl group is mono- or disubstituted with moieties            independently selected from the group consisting of:            -   (i) CONH₂ and            -   (ii) OH,    -   or R² and R³ together with the nitrogen atom between them form a        piperazine ring; and    -   R⁴ is:        -   (A) cyano,        -   (B) pyrimidine which is mono- or disubstituted with NH₂ or        -   (C) trifluoromethoxy.

Preferred compounds of the invention are those of the formula I,wherein:

-   -   R¹ is a methyl group;    -   R² and R³ are each, independently selected from the group        consisting of:        -   (A) hydrogen and        -   (B) straight or branched alkyl of 1 to 4 carbon atoms which            is mono- or disubstituted with moieties independently            selected from the group consisting of:            -   (i) CONH₂ and            -   (ii) OH; and    -   R⁴ is:        -   (A) cyano or        -   (B) trifluoromethoxy.

More preferred are compounds of the formula I wherein:

-   -   R¹ is a methyl group;    -   R² and R³ are each, independently selected from the group        consisting of:        -   (A) hydrogen and        -   (B) straight or branched alkyl of 1 to 4 carbon atoms which            is mono- or disubstituted with moieties independently            selected from the group consisting of:            -   (i) CONH₂ and            -   (ii) OH; and    -   R⁴ is trifluoromethoxy.

It will be appreciated that the compounds of the formula I have at leasttwo chiral centers. In an ultimately preferred generic aspect, theinvention includes compounds of formula I with the absolutestereochemistry depicted below in formula I*.

Specifically preferred are compounds of the formula I selected from thegroup consisting of:

(S)-2-[(R)-5-(4-Cyano-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-propionamide;(S)-2-[(R)-7-(3,5-Dichloro-phenyl)-5-methyl-6-oxo-5-(4-trifluoromethoxy-benzyl)-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-N-(2-hydroxy-2-methyl-propyl)-propionamide;(S)-2-[(R)-7-(3,5-Dichloro-phenyl)-5-methyl-6-oxo-5-(4-trifluoromethoxy-benzyl)-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-propionamide;and(S)-N-Carbamoylmethyl-2-[(R)-5-(4-cyano-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-propionamide.The invention also includes pharmaceutically acceptable salts of thecompounds of the formula I.

General Synthetic Methods

Compounds of the invention may be prepared by the general methodsdescribed below. Typically, reaction progress may be monitored by thinlayer chromatography (TLC) if desired. If desired, intermediates andproducts may be purified by chromatography on silica gel and/orrecrystallization, and characterized by one or more of the followingtechniques: NMR, mass spectroscopy and melting point. Starting materialsand reagents are either commercially available or may be prepared by oneskilled in the art using methods described in the chemical literature.

Compounds of formula I may be prepared from intermediate II. Thesynthesis of intermediate II is reported by Wu et al., U.S.Non-provisional application Ser. No. 09/604,312 and Frutos et al., U.S.Pat. No. 6,441,183, both incorporated herein by reference.

Intermediate II may be prepared by the procedure illustrated in SchemeI.

As illustrated above, intermediate III is deprotonated with a suitablebase such as lithium bis(trimethylsilyl)amide at about −20° C. to −30°C., and then alkylated with a substituted benzyl halide, preferably abenzyl bromide (IV) to produce V. Hydrolysis of the trifluoroacetamidegroup of V, for example by treatment with 40% aqueousbenzyltrimethylammonium hydroxide in dioxane/50% NaOH, followed bytreatment with acid, such as HCl, provides VI. Treatment of VI withthiocarbonyldiimidazole in the presence of a base such as4-(N,N-dimethylamino)pyridine provides VII. Treatment of VII withaminoacetaldehyde dimethyacetal and t-butylhydroperoxide solution,followed by treatment of the intermediate acetal with an acid such asp-toluenesulfonic acid provides VIII. Iodination of VIII by treatmentwith an iodinating agent such as N-iodosuccinamide provides II.

The method used for preparation of intermediate III, treatment of theamide formed from N-Boc-D-alanine and 3,5-dichloroaniline withtrifluoroacetic acid to remove the Boc-group, followed by treatment withpivalaldehyde, and acylation of the resulting imidazolodone withtrifluoroacetic anhydride is described in U.S. Pat. No. 6,414,161, citedabove, and in the chemical literature (N. Yee, Org Lett., 2000, 2,2781).

The synthesis of compounds of formula I from intermediate II isillustrated in Scheme II.

As illustrated above, treatment of II with a Grignard reagent, such ascyclopentyl magnesium bromide or chloride, followed by treatment of theresulting magnesium salt with SO₂ and then N-chlorosuccinimide providesthe sulfonyl chloride IX. Treatment of IX with the desired amine (X) inthe presence of a suitable base such as triethylamine, provides thedesired product of formula (I). Intermediates X are either commerciallyavailable or readily prepared from commercially available startingmaterials by methods known in the art. The initial product of formula Imay be further modified by methods known in the art to provideadditional compounds of the invention. Several examples are provided inthe Synthetic Examples section.

The desired R⁴ on formula I compounds may be obtained by selection ofthe appropriately substituted intermediate IV in Scheme I. Alternately,intermediate VIII having R⁴ being Br (VIIIa) may be converted tointermediates having R⁴ being CN or a substituted 5-pyrimidyl group asillustrated in Scheme III.

As illustrated above, the aryl bromide VIIIa is treated with a cyanidesalt, preferably CuCN and heated in a suitable solvent such as DMF toprovide the cyano-intermediate VIIIb. Treatment of VIIIa with apyrimidine boronate ester such as5-(4,4,5,5,-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrimidine in thepresence of a palladium catalyst such as[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)•CH₂Cl₂(PdCl₂(dppf)•CH₂Cl₂) and a base such as potassium carbonate in asuitable solvent (Suzuki reaction), for example dimethoxyethane,provides the pyrimidine intermediate VIIIc. Intermediates VIIIb andVIIIc may then be converted to desired compounds of formula I by theprocedures described in Schemes I and II. The Suzuki reaction to convertR⁴=Br to R⁴=an optionally substituted pyrimidine may also be carried outon a compound of formula I. The Suzuki reaction may also be carried outin the reverse manner. The bromide VIIIa (or a compound of formula Iwith R⁴=Br) may be converted to a boronate ester for example bytreatment with bis(pinacolato)diboron in the presence of a palladiumcatalyst such as PdCl₂(dppf) and then reacted with the desired pyrimidylbromide.

The invention is further described by the following synthetic examples.

SYNTHETIC EXAMPLES Example 1 Synthesis of(R)-5-(4-bromo-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylchloride

A solution of(R)-3-(4-bromo-benzyl)-1-(3,5-dichloro-phenyl)-3-methyl-1H-imidazo[1,2-a]imidazol-2-onein THF (0.12M) was treated with N-iodosuccinimide (1.05 equiv) andpyridinium p-toluenesulfonate (0.1 equiv). The mixture was stirred atroom temperature for 17 h, then diluted with EtOAc and washed with 10%Na₂S₂O₃ solution and water. The combined aqueous layers were extractedwith EtOAc. The combined organic phases were washed with brine, driedover Na₂SO₄, filtered and concentrated. The crude oil was purified bysilica gel chromatography to provide the desired iodide.

A solution of the above iodide in THF (0.12M) was cooled at −40° C. asc-pentyl magnesium chloride (1.05 equiv) was added dropwise over 10 min.After stirring at −40° C. for 1 h, SO₂ (g) was added by placing an inletneedle just above the surface of the reaction mixture for 1.5 min. Thebright yellow mixture was warmed to −20° C. over 1 h and then stirred atroom temperature for 1 h. N₂ (g) was bubbled through the mixture for 20min followed by concentration and pumping under high vacuum for 12 h.The resulting yellow foam was dissolved in THF (0.1M) and cooled at −20°C. as a solution of N-chlorosuccinimide (1.2 equiv) in THF (0.3M) wasadded dropwise over 5 min. After stirring at −20° C. for 1 h, themixture was poured over ice and extracted with two portions of EtOAc.The combined organic layers were washed with ice-cold brine, dried overNa₂SO₄, filtered and concentrated. Purification by silica gelchromatography provided(R)-5-(4-Bromo-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylchloride as a solid.

Example 2(S)-2-[(R)-5-[4-(4-amino-pyrimidin-5-yl)-benzyl]-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-N-(2-hydroxy-ethyl)-propionamide(660.4, M+1):

To a suspension of the hydrochloride salt of(S)-2-amino-N-(2-hydroxy-ethyl)-propionamide (0.53 g, 3.2 mmol, 3 equiv)in 10 mL of DMF was added DMAP (4 equiv), and the resulting mixture wasstirred at room temp for 1 h. To this solution was added(R)-5-(4-Bromo-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylchloride (0.580 mg, 1.06 mmol) in 2 mL of DMF at room temp. Afterstirring at room temp for 15 min, the mixture was dissolved in EtOAc andwashed with dilute water, HCl, saturated NaHCO3, and brine, dried overNa₂SO₄, filtered and concentrated. Purification of the crude product bysilica gel chromatography gave 0.305 mf of the desired product.

A mixture of(S)-2-[(R)-5-(4-bromo-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-N-(2-hydroxy-ethyl)-propionamide(0.310 g, 0.473 mmol), bis(pinacolato)diboron (0.240 g, 0.946 mmol), andKOAc (0.140 g, 1.419 mmol) in 29 mL of dioxane was flushed with N₂ for15 min. PdCl₂(dppf) (0.039 g, 0.047 mmol) was added, and the reactionmixture was heated at 80° C. for 36 h. After cooling to roomtemperature, the mixture was concentrated, and the residue was dilutedwith 150 mL of EtOAc, washed with water and brine, dried over MgSO₄, andconcentrated. The residue was purified by silica gel chromatography togive 0.204 g (62%) of the boronate.

A mixture of the above boronate (0.204 g, 0.295 mmol),5-bromo-2-amino-pyrimidine (0.078 g, 0.443 mmol), and K₂CO₃ (0.122 g,0.885 mmol) in 8 mL of dimethoxyethane and 1.2 mL of water was flushedwith N₂ for 20 min. PdCl₂(dppf) (0.025 g, 0.030 mmol) was added, and thereaction mixture was heated at 80° C. for 2 h. After cooling to roomtemperature, the mixture was diluted with EtOAc, washed with water andbrine, and concentrated. The residue was purified by silica gelchromatography and preparative TLC to afford 0.062 g (32%) of the titlecompound (660.4, M+1): The following compound was prepared by proceduresanalogous to those described in Example 2:

(S)-2-[(R)-5-[4-(4-Amino-pyrimidin-5-yl)-benzyl]-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-N-(2-hydroxy-2-methyl-propyl)-propionamide(687.1, M+1):

Example 3 Synthesis of(S)-2-[(R)-5-(4-cyano-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-propionamide

To a solution of(R)-3-(4-bromo-benzyl)-1-(3,5-dichloro-phenyl)-3-methyl-1H-imidazo[1,2-a]imidazol-2-one(3.0 g, 6.6 mmol) in DMF (60 mL) was added Zn(CN₂) (0.47 g, 4.0 mmol).The resulting solution was degassed with a strong stream of N₂ for 2 h.Pd₂dba₃ and dppf were added and the reaction mixture was dheated to 120°C. for 2 h. The solvent was evaporated and the residue dissolve inEtOAc, then was washed with water and brine, then was dried and filteredand the residue was purified over Florisil to afford 2.42 g of thedesired product. This product was then treated in a manner similar tothat described in Example 1 to produce(R)-5-(4-cyano-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylchloride.

L-Alaninamide hydrochloride (0.151 g, 1.209 mmol) was dissolved inanhydrous DMF and DMAP (0.197 g, 1.612 mmol) was added to the solution.The reaction mixture was stirred at room temperature for 1 h. Then,(R)-5-(4-cyano-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylchloride (0.24 g, 0.484 mmol) in anhydrous DMF was added to the reactionmixture and stirred for additional 10 min. The reaction solution wasdiluted with EtOAc and washed with water, 1 N HCl and then water. Theorganic phase was dried over Na₂SO₄ and concentrated under reducedpressure. The crude product was purified by silica gel columnchromatography to afford 0.211 g of the title compound as a white scalysolid (M+1, 547.2).

Example 4 Synthesis of(S)-N-carbamoylmethyl-2-[(R)-5-(4-cyano-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-propionamide

L-Alanine t-butyl ester hydrochloride (0.88 g, 4.84 mmol) was dissolvedin anhydrous DMF (10 mL) and DMAP (0.79 g, 6.46 mmol) was added to thesolution. The reaction mixture was stirred at room temperature for 1 hand(R)-5-(4-cyano-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylchloride (0.80 g, 1.61 mmol) in DMF was added to the reaction mixture.The reaction mixture was stirred at room temperature for another 10 min.The reaction mixture was washed with water (×3), 1N HCl and saturatedNaHCO₃. The organic phase was dried over Na₂SO₄ and concentrated. Thecrude product was purified by silica gel column chromatography usingCH₂Cl₂—MeOH (98:2) as an eluent to afford 0.94 g of the sulfonamidet-butyl ester. The resulting product was treated with trifluoroaceticacid (5 mL) in CH₂Cl₂ (10 mL) at room temperature for 3 h. The reactionsolution was diluted with CH₂Cl₂ and washed with water. The organicphase was dried over Na₂SO₄ and then concentrated to afford 0.67 g of(S)-2-[(R)-5-(4-cyano-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-propionicacid as a white foam.

The above carboxylic acid (0.05 g, 0.091 mmol) was dissolved inanhydrous DMF (2 mL) andbenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate(PyBOP) (0.071 g, 0.137 mmol) was added to the reaction solution. Thereaction mixture was stirred at room temperature for 10 min andglycinamide hydrochloride (0.015 g, 0.137 mmol) was added to the mixturefollowed by N,N-diisopropylethylamine (0.039 mL, 0.227 mmol). Thereaction mixture was stirred at room temperature for another 15 min. Themixture was then diluted with EtOAc (10 μL) and washed with water (×2),1N HCl, saturated NaHCO₃ and then water (×1). The organic phase wasdried over Na₂SO₄ and concentrated. The crude product was purified bysilica gel preparative thin layer chromatography using CH₂Cl₂—MeOH(95:5) as an eluent to afford 0.043 g of the title compound as a whitefoam (M+1, 604.2).

The following compound was made by procedures analogous to thosedescribed in the above example:

(S)-2-[(R)-5-(4-Cyano-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-N-(2-hydroxy-2-methyl-propyl)-propionamide:(M+1, 619)

Example 5 Synthesis of(S)-N-((R)-1-carbamoyl-ethyl)-2-[(R)-5-(4-cyano-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-propionamide

(S)-2-[(R)-5-(4-Cyano-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-propionicacid (see Example 14) (0.05 g, 0.091 mmol) was dissolved in anhydrousDMF (2 mL) and TBTU (0.044 g, 0.137 mmol) was added to the reactionsolution. The reaction mixture was stirred at room temperature for 10min and D-alaninamide hydrochloride (0.017 g, 0.137 mmol) was added tothe mixture followed by N,N-diisopropylethylamine (0.039 mL, 0.227mmol). The reaction mixture was stirred at room temperature for another15 min. The mixture was then diluted with EtOAc (10 mL) and washed withwater, 1 N HCl, saturated NaHCO₃ and then water. The organic phase wasdried over Na₂SO₄ and concentrated. The crude product was purified bysilica gel preparative thin layer chromatography to afford 0.035 g ofthe title compound as a white foam (M+1, 618.2).

Example 6(S)-2-[(R)-5-(4-cyano-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-N-(2-hydroxy-ethyl)-propionamide

A mixture of L-Boc-alanine (0.40 g, 2.11 mmol), ethanolamine (0.15 mL,2.54 mmol) and HOBt (0.29 g, 2.11 mmol) in anhydrous CH₃CN (9 mL) wascooled to 0° C. and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (EDC) (0.49 g, 2.54 mmol) was added to the reactionmixture. The reaction mixture was allowed to warm to room temperatureand stirred for 19 h. The reaction mixture was diluted with EtOAc andwashed with 5% citric acid. The aqueous layer was extracted with EtOAc.The combined organic phases were washed with saturated NaHCO₃ and brine.The organic phase was dried over Na₂SO₄ and concentrated to afford 0.38g of the coupled product as a colorless oil.

The above alcohol (0.34 g, 1.45 mmol) was dissolved in CH₂Cl₂ (3 mL) and4 N HCl in dioxane (3 ml) was added to the solution. The reactionsolution was stirred at room temperature for 2 h and then concentratedto afford (S)-2-amino-N-(2-hydroxy-ethyl)-propionamide hydrochloride.

To a solution of above amine salt (0.043 g, 0.254 mmol) in anhydrousDMF, was added DMAP (0.037 g, 0.303 mmol). The reaction mixture wasstirred at room temperature for 1 h.(R)-5-(4-cyano-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylchloride (0.036 g, 0.073 mmol) was then added to the reaction mixtureand stirred for another 2 h. The reaction mixture was diluted with EtOAcand washed with 1 N HCl and saturated NaHCO₃. The organic phase wasdried over Na₂SO₄ and concentrated. The residue was purified by silicagel preparative thin layer chromatography to afford 0.035 g of the titlecompound as a white foam (M+1, 591.1).

Example 7(S)-2-[(R)-5-(4-cyano-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-5-morpholin-4-yl-5-oxo-pentanoicacid amide

To a mixture of (S)-2-tert-butoxycarbonylamino-pentanedioic acid1-benzyl ester (4.5 g, 13.4 mmol), morpholine (1.29 mL, 14.8 mmol), HOBt(1.89 g, 14.0 mmol) in CH₂Cl₂ (18 mL) at 0° C. was slowly added asolution of DCC (2.89 g, 14.0 mmol) in CH₂Cl₂ (18 mL). The resultingmixture was allowed to warm to room temperature and was stirredovernight. The resulting precipitate was filtered off and then thefiltrate was diluted with CH₂Cl₂. The solution was washed with saturatedNaHCO₃, 1 N HCl, and finally brine. The organic layer was dried overNa₂SO₄ and then filtered. Evaporation of the solvent afforded thedesired amide (5.42 g) as a white solid.

A mixture of the above amide (6.17 g, 152 mmol) and 10% Pd/C (0.52 g) inEtOAc (50 mL) was hydrogenated under atmospheric pressure for 24 h. Themixture was filtered and concentrated to provide(S)-2-tert-butoxycarbonylamino-5-morpholin-4-yl-5-oxo-pentanoic acid(3.12 g) as a foam which was used without further purification.

Ammonia gas was bubbled into a solution of the carboxylic acid obtainedabove (3.12 g, 9.86 mmol) and O-(7-azabenzotriazol-1-yl)-N, N, N′,N′,-tetramethyluronium hexafluorophosphate (HATU) (5.25 g, 13.8 mmol) inDMF (30 mL) with stirring for 20 minutes. To the resulting yellowsuspension was added N,N-diisopropylethylamine (5.15 mL, 29.5 mmol) viasyringe. The mixture was stirred under a nitrogen atmosphere overnight.The reaction was filtered and concentrated under vacuum. The resultingresidue was dissolved in CH₂Cl₂, was washed with saturated NaHCO₃, 1 NHCl and finally brine. The organic layer was dried over Na₂SO₄, filteredand concentrated. The resulting residue was recrystallized from EtOAc toprovide the amide (1.16 g) as white solid.

The Boc group was removed with HCl in EtOAc and the resulting(S)-2-amino-5-morpholin-4-yl-5-oxo-pentanoic acid amide hydrochloridewas collected by vacuum filtration and used without furtherpurification. To a stirred solution of(R)-5-(4-cyano-benzyl)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylchloride (76 mg, 0.13 mmol) in DMF (4 mL) was addedN,N-diisopropylethylamine (60 μL, 0.39 mmol) and the amine hydrochloride(100 mg, 0.4 mmol). After stirring overnight the DMF was removed undervacuum and the resulting residue was chromatographed over silica gel.The product was then purified by semi-preparative HPLC to provide 32 mgof the title compound as a white solid (M+1, 674.04).

Example 8 Synthesis of(S)-2-[(R)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-5-(4-trifluoromethoxy-benzyl)-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-propionamide

Lithium bis(trimethylsilyl)amide (LiHMDS) (38.0 mL, 1 M in THF) wasadded slowly dropwise over 25 min to a solution of(2S,5R)-2-tert-butyl-3-(3,5-dichloro-phenyl)-5-methyl-1-(2,2,2-trifluoro-acetyl)-imidazolidin-4-one(10.0 g, 25.17 mmol) in 60 mL of THF at −20° C. After stirring at −20°C. for 20 min, a solution of 4-trifluoromethoxybenzyl bromide (6.04 mL,37.76 mmol) in 30 mL of THF was added dropwise over 20 min. The mixturewas stirred at −20° C. for 45 min, warmed to −5° C. over 1 h, and thenpoured over 50 mL of ice-cold saturated NH₄Cl solution. The resultingmixture was extracted with EtOAc. The combined organic phases werewashed with brine, dried over Na₂SO₄, filtered and concentrated. Thecrude product was triturated with hexanes to afford 12.5 g (87%) of(2R,5R)-2-tert-butyl-3-(3,5-dichloro-phenyl)-5-methyl-1-(2,2,2-trifluoro-acetyl)-5-(4-trifluoromethoxy-benzyl)-imidazolidin-4-oneas an off-white solid.

To a solution of the above imidazolidinone (6.0 g, 10.5 mmol) in 40 mLof dioxane was added 40% aqueous benzyltrimethylammonium hydroxide (6.59g, 15.75 mmol) at room temperature. As the mixture was warmed to 40° C.,50% aqueous sodium hydroxide (1.68 g, 21.0 mmol) was added slowlydropwise over 5 min. The mixture was stirred at 40° C. for 18 h, then asolution of 6.4 g of conc HCl in 3.3 mL of water was added slowlydropwise over 10 min. The mixture was warmed to 50° C. and stirred foran additional 5 h, then cooled to room temperature and concentrated. 50mL of toluene was added to the residue, and the biphasic mixture wasstirred vigorously as 50% aqueous sodium hydroxide (3.0 g) was addedslowly dropwise (pH of the aqueous phase≧10). The aqueous layer wasextracted with toluene, and the combined organic phases were washed withwater and brine, dried over Na₂SO₄, filtered and concentrated to afford4.24 g of(R)-2-amino-N-(3,5-dichloro-phenyl)-2-methyl-3-(4-trifluoromethoxy-phenyl)-propionamideas a light brown oil.

To a solution of the above propionamide (4.24 g, 10.41 mmol) in 30 mL ofTHF was added thiocarbonyldiimidazole (2.81 g, 15.77 mmol) and4-dimethylaminopyridine (DMAP) (0.127 g, 1.04 mmol). The mixture washeated at reflux for 17 h, cooled to room temperature, and concentrated.The orange oily residue was dissolved in 50 mL of toluene and treatedslowly dropwise with 20 mL of 5% aqueous HCl solution. After stirringthe mixture for 10 min, the aqueous layer was separated and extractedwith toluene. The combined organic phases were washed with water andbrine, dried over Na₂SO₄, filtered and concentrated to provide 4.48 g of(R)-3-(3,5-dichloro-phenyl)-5-methyl-2-thioxo-5-(4-trifluoromethoxy-benzyl)-imidazolidin-4-oneas an orange foam.

To a solution of the above thiohydantoin (4.47 g, 9.95 mmol) andaminoacetaldehyde dimethylacetal (6.50 mL, 59.7 mmol) in 20 mL of MeOHwas added 7.69 mL (59.7 mmol, 70% in water) of t-butyl hydroperoxidesolution, dropwise over 25 min. During the addition and for about 1 hafter, the internal temperature of the mixture was kept below 20° C.with an ice water bath. The mixture was stirred at room temperature for86 h, and 25 mL of saturated NaHSO₃ solution was added slowly dropwise,maintaining the internal temperature below 20° C. with an ice waterbath. The resulting cloudy white mixture was concentrated. To theresidue was added EtOAc, and this mixture was concentrated again. Theoily residue was partitioned between EtOAc and water, and the aqueousphase was separated and extracted with EtOAc. The combined organiclayers were washed with water and brine, dried over Na₂SO₄, filtered andconcentrated to give 5.21 g of(R)-3-(3,5-dichloro-phenyl)-2-[(E)-2,2-dimethoxy-ethylimino]-5-methyl-5-(4-trifluoromethoxy-benzyl)-imidazolidin-4-oneas a thick yellow oil.

A solution of the above crude acetal (5.20 g, 9.95 mmol) in 30 mL ofacetone was treated with p-toluenesulfonic acid (1.89 g, 9.96 mmol). Themixture was heated at reflux for 2 h, then cooled to room temperatureand concentrated. The resulting dark orange oil was dissolved in 40 mLof EtOAc and treated carefully with a solution of 2.3 g of NaHCO₃ in 23mL of water. After gas evolution ceased, the aqueous phase was separatedand extracted with two portions of EtOAc. The combined organic layerswere washed with saturated NaHCO₃ solution, two portions of water, andbrine, dried over Na₂SO₄, filtered and concentrated. The oily residuewas purified by silica gel chromatography to afford 1.58 g of(R)-1-(3,5-dichloro-phenyl)-3-methyl-3-(4-trifluoromethoxy-benzyl)-1H-imidazo[1,2-a]imidazol-2-one(456.2, M+1).

A solution of(R)-1-(3,5-dichloro-phenyl)-3-methyl-3-(4-trifluoromethoxy-benzyl)-1H-imidazo[1,2-a]imidazol-2-one(Example 1) (1.54 g, 3.38 mmol) in 30 mL of THF was treated withN-iodosuccinimide (0.846 g, 3.76 mmol) and pyridinium p-toluenesulfonate(0.086 g, 3.76 mmol). The mixture was stirred at room temperature for 17h, then diluted with EtOAc and washed with 10% Na₂S₂O₃ solution andwater. The combined aqueous layers were extracted with 10 mL of EtOAc.The combined organic phases were washed with 25 mL of brine, dried overNa₂SO₄, filtered and concentrated. The crude orange oil was purified bysilica gel chromatography to provide 1.27 g (65%) of(R)-1-(3,5-dichloro-phenyl)-5-iodo-3-methyl-3-(4-trifluoromethoxy-benzyl)-1H-imidazo[1,2-a]imidazol-2-oneas an off-white oil (582.0, M+1).

A solution of the above iodide (1.24 g, 2.13 mmol) in 16 mL of THF wascooled at −40° C. as cyclopentyl magnesium chloride (1.17 mL, 2 M indiethyl ether) was added dropwise over 10 min. After stirring at −40° C.for 1 h, SO₂ (g) was added by placing an inlet needle just above thesurface of the reaction mixture for 1.5 min. The bright yellow mixturewas warmed to −20° C. over 1 h and then stirred at room temperature for1 h. N₂ (g) was bubbled through the mixture for 20 min followed byconcentration and pumping under high vacuum for 12 h. The resultingyellow foam was dissolved in 16 mL of THF and cooled at −20° C. as asolution of N-chlorosuccinimide (0.341 g, 2.56 mmol) in 8 mL of THF wasadded dropwise over 5 min. After stirring at −20° C. for 1 h, themixture was poured over ice and extracted with two portions of EtOAc.The combined organic layers were washed with ice-cold brine, dried overNa₂SO₄, filtered and concentrated. Purification by silica gelchromatography provided 0.975 g (83%) of(R)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-5-(4-trifluoromethoxy-benzyl)-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylchloride thick oil (554.2, M+1).

A solution of L-alaninamide HCl salt (0.097 g, 0.782 mmol) in 6.5 mL ofDMF was treated with triethylamine (0.163 mL, 1.17 mmol) at roomtemperature. After stirring for 10 min, a solution of the above sulfonylchloride (0.217 g, 0.391 mmol) in 1 mL of CH₂Cl₂ was added rapidlydropwise, and the cloudy mixture was stirred at room temperature for 5h. Following the addition of EtOAc, the organic layer was washed withwater, then brine, dried over Na₂SO₄, filtered and concentrated. Thecrude product was purified by silica gel chromatography to afford 0.193g (81%) of the title compound as a white solid (606.3, M+1).

The following compound was prepared by a procedure analogous to Example8:

(R)-2-[(R)-7-(3,5-Dichloro-phenyl)-5-methyl-6-oxo-5-(4-trifluoromethoxy-benzyl)-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-propionamide(606.4, M+1):

Example 9 Synthesis of(S)-2-[(R)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-5-(4-trifluoromethoxy-benzyl)-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-N-(2-hydroxy-ethyl)-propionamide.

To a suspension of N-Boc-L-(N-hydroxyethyl)alaninamide (0.188 g, 0.809mmol) in 1 mL of dioxane was added HCl (2.0 mL, 4 M in dioxane), and theresulting cloudy mixture was stirred at room temperature for 2.5 h.Concentration of the mixture was followed by addition of CH₂Cl₂, andthis process was repeated twice. Final pumping under high vacuum for 12h afforded a colorless oil. The crude amine HCl salt was dissolved in1.5 mL of DMF and treated with triethylamine (0.157 mL, 1.13 mmol).After stirring at room temperature for 10 min, a solution of(R)-7-(3,5-dichloro-phenyl)-5-methyl-6-oxo-5-(4-trifluoromethoxy-benzyl)-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylchloride (0.125 g, 0.225 mmol) in 3 mL of CH₂Cl₂ was added rapidlydropwise via cannula. The reaction mixture was stirred at roomtemperature for 4 h. Following the addition of EtOAc, the organic layerwas washed with three portions of 5% NaCl solution, then brine, driedover Na₂SO₄, filtered and concentrated. The crude product was purifiedby preparative TLC to afford 0.118 g (81%) of the title compound as awhite foam (649.9, M+1).

The following compound was prepared by a procedure analogous to Example9:

(S)-2-[(R)-7-(3,5-Dichloro-phenyl)-5-methyl-6-oxo-5-(4-trifluoromethoxy-benzyl)-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonylamino]-N-(2-hydroxy-2-methyl-propyl)-propionamide(678.3, M+1):

Description of Biological Properties

The biological properties of representative compounds of the formula Iwere investigated by way of the experimental protocol described below.

Assay to Determine Inhibition of LFA-1 Binding to ICAM-1

Purpose of Assay:

This assay protocol is designed to study the direct antagonism, by atest compound, of the interaction of the CAM, ICAM-1 with theLeukointegrin CD18/CD11a (LFA-1).

Description of Assay Protocol:

LFA-1 is immunopurified using the TS2/4 antibody from a 20 g pellet ofhuman JY or SKW3 cells, utilizing a protocol previously described(Dustin, M. J.; et al., J. Immunol. 1992, 148, 2654-2660). The LFA-1 ispurified from SKW3 lysates by immunoaffinity chromatography on TS2/4LFA-1 mAb Sepharose and eluted at pH 11.5 in the presence of 2 mM MgCl₂and 1% octylglucoside. After collection and neutralization of fractionsfrom the TS2/4 column, samples are pooled and precleared with Protein Gagarose.

A soluble form of ICAM-1 is constructed, expressed, purified andcharacterized as previously described (Marlin, S.; et al., Nature, 1990,344, 70-72 and see Arruda, A.; et al., Antimicrob. Agents Chemother.1992, 36, 1186-1192). Briefly, isoleucine 454 which is located at theputative boundary between domain 5 of the ectodomain and thetransmembrane domain, is changed to a stop codon using standardoligonucleotide-directed mutagenesis. This construction yields amolecule identical with the first 453 amino acids of membrane boundICAM-1. An expression vector is created with a hamster dihydrofolatereductase gene, a neomycin-resistance marker, and the coding region ofthe sICAM-1 construct described above, along with the promoter, splicesignals, and polyadenylation signal of the SV40 early region. Therecombinant plasmid is transfected into CHO DUX cells using standardcalcium phosphate methods. Cells are passaged in selective media (G418)and colonies secreting sICAM-1 are amplified using methotrexate. sICAM-1is purified from serum-free media using traditional non-affinitychromatographic techniques, including ion exchange and size exclusionchromatography.

LFA-1 binding to ICAM-1 is monitored by first incubating sICAM-1 at 40μg/mL in Dulbecco's phosphate buffered saline with calcium andmagnesium, additional 2 mM MgCl₂ and 0.1 mM PMSF (Diluting Buffer) in a96-well plate for 30 min at room temperature. Plates are then blocked bythe addition of 2% (w/v) bovine serum albumin in Diluting Buffer for 37°C. for 1 h. Blocking solution is removed from wells, and test compoundsare diluted and then added followed by the addition of approximately 25ng of immunoaffinity purified LFA-1. The LFA-1 is incubated in thepresence of test compound and ICAM-1 at 37° C. for 1 h. Wells are washed3 times with Diluting Buffer. The bound LFA-1 is detected by theaddition of a polyclonal antibody directed against a peptidecorresponding to the CD18 cytoplasmic tail in a 1:100 dilution withDiluting Buffer and 1% BSA and allowed to incubate for 45 min at 37° C.Wells are washed 3 times with Diluting Buffer and the bound polyclonalantibody is detected by the addition of a 1:4000 dilution of horseradish peroxidase conjugated to goat immunoglobulin directed againstrabbit immunoglobulin. This reagent is allowed to incubate for 20 min at37° C., wells are washed as above and the substrate for the horse radishperoxidase is added to each well to develop a quantitative colorimetricsignal proportional to the amount of LFA-1 bound to sICAM-1. SolubleICAM-1 (60 μg/mL) is used as a positive control for inhibition of theLFA-1/ICAM-1 interaction. The lack of the addition of LFA-1 to thebinding assay is used as a background control for all samples. Adose-response curve is obtained for all test compounds.

All compounds made in the above examples were tested in this assay andeach found to have a K_(d)<10 μM.

Assay to Determine Metabolism by Human Liver Microsomal Enzymes

Purpose of Assay:

This assay protocol is designed to measure the in vitro metabolism oftest compounds by human liver microsomal enzymes. The data collected areanalyzed to calculate a half-life (t_(1/2), min) for test compounds.

Description of Assay Protocol:

The assay is performed in 50 mM potassium phosphate buffer, pH 7.4 and2.5 mM NADPH. Test samples are dissolved in acetonitrile for a finalassay concentration of 1-10 μM. Human liver microsomes are diluted inassay buffer to a final assay concentration of 1 mg protein/mL. A volumeof 25 μL compound solution and 50 μL microsome suspension are added to825 μL assay buffer. The preparation is incubated for 5 min in a 37° C.water bath. The reaction is started by the addition of 100 μL NADPH.Volumes of 80 μL are removed from the incubation mix at 0, 3, 6, 10, 15,20, 40, and 60 min after the start of the reaction and added to 160 μLacetonitrile. The samples are shaken for 20 sec and then centrifuged for3 min at 3000 rpm. A 200 μL volume of the supernatant is transferred to0.25 mm glass fiber filter plates and centrifuged for 5 min at 3000 rpm.Injection volumes of 10 μL are typically added to Zorbax SB C8 HPLCcolumns with formic acid in water or acetonitrile at a flow rate of 1.5mL/min. Percent loss of parent compound is calculated from the areaunder each time point to determine the half-life.

Compounds made in the above examples were tested in this assay andgenerally found to have a t_(1/2)≧50 minutes.

Description of Therapeutic Use

The novel small molecules of formula I provided by the invention inhibitthe ICAM-1/LFA-1 dependent homotypic aggregation of human lymphocytesand human lymphocyte adherence to ICAM-1. These compounds havetherapeutic utility in the modulation of immune cellactivation/proliferation, e.g., as competitive inhibitors ofintercellular ligand/receptor binding reactions involving CAMs andLeukointegrins. To be more specific, the compounds of the invention maybe used to treat certain inflammatory conditions, including conditionsresulting from a response of the non-specific immune system in a mammal(e.g., adult respiratory distress syndrome, shock, oxygen toxicity,multiple organ injury syndrome secondary to septicemia, multiple organinjury syndrome secondary to trauma, reperfusion injury of tissue due tocardiopulmonary bypass, myocardial infarction or use with thrombolysisagents, acute glomerulonephritis, vasculitis, reactive arthritis,dermatosis with acute inflammatory components, stroke, thermal injury,hemodialysis, leukapheresis, ulcerative colitis, necrotizingenterocolitis and granulocyte transfusion associated syndrome) andconditions resulting from a response of the specific immune system in amammal (e.g., psoriasis, organ/tissue transplant rejection, graft vs.host reactions and autoimmune diseases including Raynaud's syndrome,autoimmune thyroiditis, dermatitis, multiple sclerosis, rheumatoidarthritis, insulin-dependent diabetes mellitus, uveitis, inflammatorybowel disease including Crohn's disease and ulcerative colitis, andsystemic lupus erythematosus). The compounds of the invention may alsobe used in treating asthma or as an adjunct to minimize toxicity withcytokine therapy in the treatment of cancers. In general these compoundsmay be employed in the treatment of those diseases currently treatablethrough steroid therapy.

Thus, another aspect of the invention is the provision of a method forthe treatment or prophylaxis of the above-described conditions throughthe adminstration of therapeutic or prophylactic amounts of one or morecompounds of the formula I.

In accordance with the method provided by the invention, the novelcompounds of formula I may be administered for either a prophylactic ortherapeutic purpose either alone or with other immunosuppressive orantiinflammatory agents. When provided prophylactically, theimmunosuppressive compound(s) are provided in advance of anyinflammatory response or symptom (for example, prior to, at, or shortlyafter the time of an organ or tissue transplant but in advance of anysymptoms of organ rejection). The prophylactic administration of acompound of the formula I serves to prevent or attenuate any subsequentinflammatory response (such as, for example, rejection of a transplantedorgan or tissue, etc.). The therapeutic administration of a compound ofthe formula I serves to attenuate any actual inflammation (such as, forexample, the rejection of a transplanted organ or tissue). Thus, inaccordance with the invention, a compound of the formula I can beadministered either prior to the onset of inflammation (so as tosuppress an anticipated inflammation) or after the initiation ofinflammation.

The novel compounds of the formula I may, in accordance with theinvention, be administered in single or divided doses by the oral,parenteral or topical routes. A suitable oral dosage for a compound offormula I would be in the range of about 0.1 mg to 10 g per day. Inparenteral formulations, a suitable dosage unit may contain from 0.1 to250 mg of said compounds, whereas for topical administration,formulations containing 0.01 to 1% active ingredient are preferred. Itshould be understood, however, that the dosage administration frompatient to patient will vary and the dosage for any particular patientwill depend upon the clinician's judgement, who will use as criteria forfixing a proper dosage the size and condition of the patient as well asthe patient's response to the drug.

When the compounds of the present invention are to be administered bythe oral route, they may be administered as medicaments in the form ofpharmaceutical preparations which contain them in association with acompatible pharmaceutical carrier material. Such carrier material can bean inert organic or inorganic carrier material suitable for oraladministration. Examples of such carrier materials are water, gelatin,talc, starch, magnesium stearate, gum arabic, vegetable oils,polyalkylene-glycols, petroleum jelly and the like.

The pharmaceutical preparations can be prepared in a conventional mannerand finished dosage forms can be solid dosage forms, for example,tablets, dragees, capsules, and the like, or liquid dosage forms, forexample solutions, suspensions, emulsions and the like. Thepharmaceutical preparations may be subjected to conventionalpharmaceutical operations such as sterilization. Further, thepharmaceutical preparations may contain conventional adjuvants such aspreservatives, stabilizers, emulsifiers, flavor-improvers, wettingagents, buffers, salts for varying the osmotic pressure and the like.Solid carrier material which can be used include, for example, starch,lactose, mannitol, methyl cellulose, microcrystalline cellulose, talc,silica, dibasic calcium phosphate, and high molecular weight polymers(such as polyethylene glycol).

For parenteral use, a compound of formula I can be administered in anaqueous or non-aqueous solution, suspension or emulsion in apharmaceutically acceptable oil or a mixture of liquids, which maycontain bacteriostatic agents, antioxidants, preservatives, buffers orother solutes to render the solution isotonic with the blood, thickeningagents, suspending agents or other pharmaceutically acceptableadditives. Additives of this type include, for example, tartrate,citrate and acetate buffers, ethanol, propylene glycol, polyethyleneglycol, complex formers (such as EDTA), antioxidants (such as sodiumbisulfite, sodium metabisulfite, and ascorbic acid), high molecularweight polymers (such as liquid polyethylene oxides) for viscosityregulation and polyethylene derivatives of sorbitol anhydrides.Preservatives may also be added if necessary, such as benzoic acid,methyl or propyl paraben, benzalkonium chloride and other quaternaryammonium compounds.

The compounds of this invention may also be administered as solutionsfor nasal application and may contain in addition to the compounds ofthis invention suitable buffers, tonicity adjusters, microbialpreservatives, antioxidants and viscosity-increasing agents in anaqueous vehicle. Examples of agents used to increase viscosity arepolyvinyl alcohol, cellulose derivatives, polyvinylpyrrolidone,polysorbates or glycerin. Microbial preservatives added may includebenzalkonium chloride, thimerosal, chloro-butanol or phenylethylalcohol.

Additionally, the compounds provided by the invention can beadministered topically or by suppository.

FORMULATIONS

Compounds of the formula I can be formulated for therapeuticadministration in a number of ways. Descriptions of several exemplaryformulations are given below.

Example A

Capsules or Tablets Example A-1 Example A-2 Ingredients QuantityIngredients Quantity Compound of 250 mg Compound of formula I 50 mgformula I Starch 160 mg Dicalcium Phosphate 160 mg  Microcrys. Cellulose 90 mg Microcrys. Cellulose 90 mg Sodium Starch  10 mg Stearic acid  5mg Glycolate Magnesium Stearate  2 mg Sodium Starch Glycolate 10 mgFumed colloidal silica  1 mg Fumed colloidal silica  1 mg

The compound of formula I is blended into a powder mixture with thepremixed excipient materials as identified above with the exception ofthe lubricant. The lubricant is then blended in and the resulting blendcompressed into tablets or filled into hard gelatin capsules.

Example B

Parenteral Solutions Ingredients Quantity Compound of formula I 500 mgPEG 400 40% by volume Ethyl Alcohol 5% by volume Saline 55% by volume

The excipient materials are mixed and then added to one of the compoundsof formula I in such volume as is necessary for dissolution. Mixing iscontinued until the solution is clear. The solution then filtered intothe appropriate vials or ampoules and sterilized by autoclaving.

Example C

Suspension Ingredients Quantity Compound of formula I 100 mg Citric acid1.92 g Benzalkonium chloride 0.025% by weight EDTA 0.1% by weightPolyvinylalcohol 10% by weight Water q.s. to 100 mL

The excipient materials are mixed with the water and thereafter one ofthe compounds of formula I is added and mixing is continued until thesuspension is homogeneous. The suspension is then transferred into theappropriate vials or ampoules.

Example D

Topical Formulation Ingredients Quantity Compound of formula I   5% byweight Tefose 63   13% by weight Labrafil M 1944 CS   3% by weightParaffin Oil   8% by weight Methylparaben (MP) 0.15% by weightPropylparaben (PP) 0.05% by weight Deionized water q.s. to 100

The proper amounts of Tefose 63, Labrafil M 1944 CS, Paraffin oil andwater are mixed and heated at 75° C. until all components have melted.The mixture is then cooled to 50° C. with continuous stirring.Methylparaben and propylparaben are added with mixing and the mixture iscooled to ambient temperature. The compound of formula I is added to themixture and blended well.

1. A method for treating inflammation or an inflammatory condition in apatient which comprises administering to said patient a therapeuticallyeffective amount of a compound of the formula I:

wherein: R¹ is straight or branched alkyl of 1 to 3 carbon atoms whichis optionally mono- or disubstituted with oxo; R² and R³ are eachindependently selected from the group consisting of: (A) hydrogen, and(B) straight or branched alkyl of 1 to 4 carbon atoms which alkyl groupis mono- or disubstituted with moieties independently selected from thegroup consisting of: (i) CONH₂ and (ii) OH,  and R⁴ is: (A) cyano, or(B) trifluoromethoxy; or a pharmaceutically acceptable salt thereof. 2.The method of claim 1 wherein the condition to be treated is adultrespiratory distress syndrome, shock, oxygen toxicity, multiple organinjury syndrome secondary to septicemia, multiple organ injury syndromesecondary to trauma, reperfusion injury of tissue due to cardiopulmonarybypass, myocardial infarction or use with thrombolysis agents, acuteglomerulonephritis, vasculitis, reactive arthritis, dermatosis withacute inflammatory components, stroke, thermal injury, hemodialysis,leukapheresis, ulcerative colitis, necrotizing enterocolitis orgranulocyte transfusion associated syndrome.
 3. The method of claim 1wherein the condition to be treated is psoriasis, organ/tissuetransplant rejection, graft vs. host reaction or an autoimmune disease.4. The method of claim 1 wherein the condition to be treated ispsoriasis.
 5. The method of claim 1 wherein the condition to be treatedis Raynaud's syndrome, autoimmune thyroiditis, dermatitis, multiplesclerosis, rheumatoid arthritis, insulin-dependent diabetes mellitus,uveitis, inflammatory bowel disease, Crohn's disease, ulcerative colitisor systemic lupus erythematosus.
 6. The method of claim 1 wherein thecondition to be treated is multiple sclerosis.
 7. The method of claim 1wherein the condition to be treated is asthma.
 8. The method of claim 1wherein the condition to be treated is the toxic effects of cytokinetherapy.